Chanan‐Khan, Asher

Abstract Abstract 4919 Waldenstrom's macroglobulinemia (WM) is characterized by the presence of lymphoplasmacytic cells in the bone marrow and the secretion of IgM monoclonal antibody in the serum. Several conventional therapies are available but the disease remains incurable. Recently, bortezomib (a proteasomal inhibitor) has shown promising anti-WM activity with enhanced responses when combined with traditional therapies. Resistance to bortezomib therapy is an important event that is associated with continued treatment. In order to understand the mechanism of bortezomib resistance in WM we exposed BCWM.1 (a known WM cell line) in vitro to increasing concentrations of bortezomib over prolonged periods of time and isolated the bortezomib resistant clone (BCWM.1/BR). This clone was compared with its parent wild type cell line (BCWM.1/WT). Investigation to understand the susceptibility of BCWM.1/Br cells to various therapeutic agents showed that these cells are resistant to many of the agents such as melaphalan, fludarabine or doxorubicin. Interestingly, verapamil, a broad spectrum inhibitor of multidrug resistance, failed to reverse the resistance induced by bortezomib indicating that bortezomib resistance is not because of an activation of multidrug resistance in these cells. While BCWM.1/WT cells showed an IC50 of 7.8nM when treated for 72h with bortezomib, the BCWM.1/BR cells were not inhibited at any concentration of the compound up to 100nM. Furthermore, the cells with the acquired resistance showed a 4 fold increase in the proteasomal activity as measured by the release of a fluorescent product (7-Amino-4-methylcoumarin (AMC)) from its peptide substrate, suc-LLVY-AMC. Biochemical analysis further revealed that many of the proteasomal components are altered in BCWM.1/BR cells as compared to their parental control cells. Interestingly, protein levels of two of the proteasomal catalytic subunits, PSMB5 and PSMB9 are upregulated in resistant cells suggesting a reason for the enhanced proteasomal activity of these cells. The resistant cells showed an altered gene expression profile that indicates a transformation of the parental wild type cell line to acquire resistance. A comparative analysis of the signal transduction pathways operated in these cells showed that many of the activation and cell survival pathways that are present in BCWM.1 cells are inhibited in the resistant cells. For example, BCWM.1 cells show a constitutive activation of AKT and ERK1/2 which are inhibited in the resistant cells thus making them insensitive to the inhibitors of these pathways. Similarly, HSP27 which was earlier shown to contribute to bortezomib induced resistance was completely inhibited in BCWM.1 resistant cells. Interestingly, there is an increase in Bcl-2 protein in BCWM.1/BR cells as compared to WT cells indicating that the resistant cells might be dependent on Bcl-2 family for their survival. Inhibition of Bcl-2 induced potent apoptosis in BCWM.1/BR cells. Thus the results presented here indicate that acquired bortezomib resistance in BCWM.1 cells alters their proteasomal activity, cellular signaling pathways to make them resistant to many of the known therapies but these cells retain the Bcl-2 mediated pathway for targeting thus inhibitors of Bcl-2 may be used in therapy against bortezomib-resistant WM. Disclosures Chanan-Khan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract Abstract 1875 Introduction: Carfilzomib (CFZ) is a next-generation proteasome inhibitor that has demonstrated durable single-agent antitumor activity in patients (pts) with relapsed and/or refractory multiple myeloma (R/R MM). Previously we have shown that unfavorable cytogenetic features, defined as presence of any of del 13 or hypodiploidy by metaphase cytogenetic analysis and/or del 17p13, t(4;14), t (14;16) by fluorescence in situ hybridization (FISH), did not appreciably affect overall (≥PR) and clinical benefit (≥MR) response rates (ORR and CBR) in PX-171-003-A1, a large phase 2 study of single-agent CFZ in pts with R/R MM (Jakubowiak et al. Ann Oncol. 2011;22(S4): Abstract 117). The objectives of the present analysis were to evaluate the impact of specific cytogenetic abnormalities on efficacy and treatment outcomes. Methods: Pts with unfavorable cytogenetic abnormalities (defined above) were eligible for inclusion in this analysis. All pts received CFZ at 20 mg/m2 IV on days 1, 2, 8, 9, 15, and 16 of 28-day cycles (C) and were dose-escalated to 27 mg/m2 at C2. Pts were treated for up to 12 C. Responses were assessed by International Myeloma Working Group criteria, with minimal response (MR) by European Group for Blood and Marrow Transplantation criteria. Assessments were confirmed by an Independent Review Committee (IRC). Results: A total of 234 pts were included in this analysis. Metaphase cytogenetic data were available for 203 pts (87%), and FISH data were available for 210 pts (90%); 178 pts had both (76%). Seventy-five of 234 pts (32%) had ≥1 abnormality and represented the unfavorable group in this analysis. Of these, 49 (65%) had abnormalities detected via metaphase cytogenetics, 47 (63%) by FISH, and 21 (28%) by both methods. Characteristics of pts in the unfavorable and normal/favorable groups were comparable overall, with the exception of a higher proportion of pts with International Staging System stage II/III in the unfavorable group (83% vs 64%). Response rates per the IRC, presented previously, included an ORR of 30% for pts with ≥1 abnormality compared with 23% for pts with none. The corresponding CBRs were 34% and 40%, respectively. Duration of response (DOR) and time to progression (TTP) values were 7 months (mo) (95% CI 4–9) and 4 mo (95% CI 3–6), respectively, for pts with ≥1 abnormality and 8 mo (95% CI 6–12) and 5 mo (95% CI 3–6), respectively, for pts with none. The ORRs, CBRs, and disease control rates (DCRs) according to specific cytogenetic abnormalities are presented below. Response rates were largely unaffected by the presence of specific unfavorable cytogenetic abnormalities, with the possible exceptions of trends toward a higher response rate for t(4;14) and a lower rate for t(14;16), although the latter abnormality was only noted in 3 pts. The trend for better response rates in pts with t(4;14) corresponded to a DOR of 5.6 mo (95% CI 2.8–10.2). In contrast, the DOR appeared shorter for the subgroup of pts with del 17p13 (4.6 mo, 95% CI: 3.7–Not reached). Time-to-event endpoints including TTP are being analyzed for these and the other subpopulations, and data on these will be presented, along with overall survival stratified by cytogenetic abnormality. Conclusions: CFZ demonstrated comparable response rates in pts with relapsed/refractory MM in both the absence and presence of unfavorable cytogenetic abnormalities in the 003-A1 study. Similarly, in the phase 2 PX-171-004 study, no significant difference in response rates between unfavorable and normal/favorable groups has been observed in a population of less heavily-pretreated pts with relapsed or R/R MM. Taken together, the results of these and other analyses to date suggest that responses to single-agent CFZ can be achieved in heavily pretreated pts and that overall, these responses appear not to be significantly influenced by poor prognostic cytogenetic features. Further analyses from studies with CFZ in newly diagnosed MM and in less heavily-pretreated pts are needed to determine whether a trend toward lower response rates and DOR for pts with del 17p13 is consistently observed in other populations. Disclosures: Jakubowiak: Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria. Off Label Use: Use of the investigational agent carfilzomib, proteasome inhibitor, in the treatment of relapsed and/or refractory multiple myeloma. Siegel:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Singhal:Celgene: Speakers Bureau; Millennium: Speakers Bureau; Onyx Pharmaceuticals: Research Funding. Martin:Celgene: Honoraria; Onyx Pharmaceuticals: Consultancy. Lonial:Onyx: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Millennium Pharmaceuticals, Inc.: Consultancy; Merck: Consultancy. Kukreti:Celgene: Honoraria. Bahlis:Celgene: Honoraria, Speakers Bureau. Alsina:Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Allergan: Research Funding. Kunkel:Onyx Pharmaceuticals: Consultancy; Threshold: Consultancy; VLST biothech: Consultancy. Wong:Onyx Pharmaceuticals: Employment, Equity Ownership. Orlowski:Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Stewart:Celgene Corporation: Consultancy, Research Funding; Millenium: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jagannath:Millennium: Honoraria; Celgene: Honoraria; Onyx Pharmaceuticals: Honoraria; Merck: Honoraria; OrthoBiotec. Imedex: Membership on an entity's Board of Directors or advisory committees; Medicom World Wide: Membership on an entity's Board of Directors or advisory committees; Optum Health Education: Membership on an entity's Board of Directors or advisory committees; PER Group: Membership on an entity's Board of Directors or advisory committees. Wang:Onyx Pharmaceuticals: Research Funding.

Abstract Abstract 985 Introduction: CFZ is a novel and highly selective epoxyketone proteasome inhibitor currently in clinical development for the treatment of multiple myeloma (MM). Ph 1 and 2 studies with CFZ have demonstrated durable single-agent antitumor activity in pts with relapsed or refractory (R/R) MM. The present study, PX-171-003-A1, was an open-label, single-arm Ph 2b trial and enrolled patients with multiply relapsed MM whose disease was refractory (defined as <25% response on, or progression during or <60 days after completion of, therapy) to their last treatment regimen. Patients must have received ≥2 prior therapies including: 1) bortezomib (BTZ) and either thalidomide (THAL) or lenalidomide (LEN), and 2) an alkylating agent. Materials and Methods: Pts received CFZ at 20 mg/m2 on a QDx2 schedule (Days 1, 2, 8, 9, 15, and 16 every 28 days) in cycle (C) 1 and were dose escalated to 27 mg/m2 on the same schedule thereafter for up to 12 C. Pts completing 12 C were eligible to enter an extension study (PX-171-010). The primary endpoint was overall response rate (ORR) (≥ partial response [PR]). Secondary endpoints included: clinical benefit response (CBR) (ORR + Minimal response [MR]), duration of response for ≥PR (DOR), overall survival (OS), time to progression (TTP), progression free survival (PFS), and safety. Responses and progression were determined according to the International Myeloma Working Group (IMWG) criteria and were assessed and confirmed by an Independent Response Committee (IRC). Results: 266 pts were enrolled with a median duration of MM of 5.4 years including 83% whose disease had progressed on or within 60 d of last therapy and 17% whose disease had achieved <25 % response to the regimen immediately preceding study entry. Of the 266 pts enrolled pts, 257 were evaluable for response; 9 patients were considered not evaluable based on missing baseline or lacking at least one post-baseline M-protein. An ORR (≥PR) of 24% with a median DOR of 7.4 mo (range 6.2–10.3) was determined. Responses are detailed in the table. The CBR (ORR + MR) was 36%. Median DOR of pts with MRs was 6.3 months, indicating that long-term MRs were observed. An additional 32% (83 pts) achieved SD for at least 6 wks. To date, 79 pts (30%) completed ≥6 C and >11% of pts have completed all 12 C of protocol specified therapy and most have entered the extension protocol; 15 pts remain on study (all >10 C). OS and TTP data for the overall population will also be reported. The enrolled pts in this study were heavily pretreated having received a median of 5 prior lines of therapy (range 1–20, median of 13 anti-myeloma agents). 85% of pts had received at least 2 and 37% had received at least 3 drugs in the regimen just prior to entering the study. Prior anti-myeloma agents included 99.6% (265/266 pts) BTZ (median 2 prior regimens containing BTZ), 99.6% either THAL (74%) or LEN (94%), 98% corticosteroids, 91% alkylating agents, and 74% stem cell transplant; 65% of pts were refractory to BTZ at any point in time prior to study entry. The most common treatment-emergent adverse events ≥ Grade (G) 3 regardless of relationship to study drug were predominantly hematologic and included thrombocytopenia (22%), anemia (20%), lymphopenia (10%), pneumonia (8%), neutropenia (8%), fatigue (7%), hyponatremia (5%), and hypercalcemia (5%). Although 206 pts (77%) had G1/2 peripheral neuropathy (PN) at baseline, new onset PN was infrequent and G ≥3 PN occurred in <1%. Interestingly, in this subset of patients, efficacy response was nearly identical to that seen in the full study population with an ORR (≥PR) of 24%. Conclusions: Single-agent CFZ achieved durable responses in pts with R/R MM whose disease had relapsed after all available therapies including BTZ and immunomodulatory agents. The CBR and median DOR achieved with this steroid-sparing regimen establish that CFZ has the potential to offer substantial clinical benefit to patients with relapsed or refractory disease. CFZ was well-tolerated and side effects were clinically manageable with no new or unexpected toxicities observed. Importantly, exacerbation of pre-existing PN was uncommon. Cumulative side effects were not observed, allowing prolonged single-agent dosing for chronic disease control. Disclosures: Siegel: Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Martin:Celgene: Honoraria; Onyx: Consultancy. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Vij:Onyx: Honoraria. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Jagannath:Millenium, OrthoBiotec, Celgene, Merck, Onyx: Honoraria; Imedex, Medicom World Wide, Optum Health Education, PER Group: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Kukreti:Celgene: Honoraria; Roche: Honoraria; Ortho Biotech: Honoraria. Alsina:Millenium: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy. Zonder:Millenium: Consultancy, Honoraria, Research Funding; Cephalon: Research Funding; Celgene: Honoraria. Wong:Onyx Pharmaceuticals: Employment. Vallone:Onyx Pharmaceuticals: Employment. Chang:Onyx Pharmaceuticals: Employment. Kauffman:Onyx Pharmaceuticals: Employment. Stewart:Millennium: Consultancy; Celgene: Honoraria. Singhal:Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx : Research Funding.

Abstract Background BT062 (Biotest AG Dreieich, Germany) is an antibody-drug conjugate, comprising the anti-CD138 chimerized MAb (nBT062) and the maytansinoid DM4 as cytotoxic agent. Once bound to CD138 on a target cell, the conjugate is internalized and releases DM4, leading to target cell death. CD138 (Syndecan-1) is highly overexpressed on various solid tumors and in hematological malignancies, and represents one of the most specific target antigens for identification of multiple myeloma (MM) cells. Data from two studies investigating BT062 as single agent demonstrated an acceptable tolerability profile and evidence of clinical activity in patients with heavily pretreated relapsed and/or refractory MM (1, 2). Preclinical studies showed enhanced anti-MM activity when BT062 was combined with lenalidomide and dexamethasone (Len/Dex). Based on these data, a Phase I/IIa study in MM was initiated to evaluate the safety and efficacy of BT062 in combination with Len/Dex. Objectives To determine the dose-limiting toxicities (DLTs), the maximum tolerated dose (MTD), the recommended phase II dose (RPTD), pharmacokinetics (PK), and anti-MM activity of increasing doses of BT062 (days 1, 8, and 15, every 4 weeks) in combination with Len (25 mg, daily on days 1-21) and low dose Dex (40 mg on days 1, 8, 15, and 22) in patients with relapsed and/or refractory MM. Methods This is a prospective, open label, dose-escalation, multicenter Phase I/IIa study. The Phase I part includes dose escalation, and the Phase IIa the expansion of the MTD or RPTD cohort. Patients aged ≥18 years with relapsed and/or refractory MM who have failed at least one prior therapy were eligible to participate. Prior treatment with Len and/or Dex was allowed. Patients with clinical response (or no evidence of progressive disease) and without unacceptable toxicities were eligible for additional treatment cycles. Patients were enrolled in cohorts of at least 3 at each dose level; DLT in the first cycle triggered cohort expansion. Toxicities were assessed by CTCAE v4 and clinical response was assessed according to International Myeloma Working Group criteria. Results As of July 2013, a total of 15 patients have received BT062 at dose levels of 80 mg/m2 (N=3), 100 mg/m2 (N=6) or 120 mg/m2 (N=6). Two patients at the highest dose level discontinued study due to toxicity (DLT), another patient withdrew consent. The other 12 patients remain on treatment; median duration 144 days (range 8–385). The median number of prior therapies was 4 (range 1–11), 87% of patients had prior Len exposure, and 50% were Len/Dex refractory. The maximum administered dose (MAD) has been reached at 120 mg/m2, with mucosal inflammation (CTC grade 3) as DLT in one, and anemia (CTC grade 3) in a second of the 6 patients treated at this dose level. About 85% of reported Adverse Events (AE) were of CTC grade 1 or 2. The most common reported AEs were fatigue, hypokalemia, and diarrhea. Amongst the 9 patients currently evaluable for efficacy, responses were observed across all dose levels with a overall response rate (ORR) of 78%; including 1 patient with complete response (120 mg/m2), 1 patient with very good partial response (80 mg/m2), and 5 patients with partial response (80 and 100 mg/m2). Two other patients achieved disease stabilization, resulting in a clinical benefit in 100% of the evaluated patients. Interestingly, partial response was observed in 3 patients refractory to prior treatment with Len/Dex. The MTD has been defined as 100 mg/m2 and is currently expanded to further evaluate the safety and efficacy of BT062 at the RPTD. Conclusion Preliminary data from this ongoing study indicate that BT062 is well tolerated in combination with Len/Dex at dose levels that induce responses in patients with relapsed and/or refractory multiple myeloma, including Len/Dex-refractory patients. Updated results on safety and efficacy will be presented. References 1. Jagannath et al, BT062, an Antibody-Drug Conjugate Directed Against CD138, Shows Clinical Activity in Patients with Relapsed or Relapsed/Refractory Multiple Myeloma. Blood. 2011; 118: Abstract 305. 2. Heffner et al, BT062, an Antibody-Drug Conjugate Directed Against CD138, Given Weekly for 3 Weeks in Each 4 Week Cycle: Safety and Further Evidence of Clinical Activity. Blood. 2012; 120: Abstract 4042. Disclosures: Somlo: Celgene: Research Funding, Speakers Bureau; NIH: Research Funding; Millennium: Speakers Bureau. Heffner:Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Biotest: Honoraria, Research Funding; Onyx: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding. Siegel:Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Zimmerman:Celgene: Honoraria; Millennium: Honoraria; Onyx: Honoraria. Jagannath:Millennium: Honoraria; Celgene: Honoraria. Munshi:Celgene: Consultancy; Novartis: Consultancy; Millennium: Consultancy. Lonial:Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy. Ruehle:Biotest AG: Employment. Chavan:Biotest Pharmaceuticals: Employment. Patel:Biotest Pharmaceuticals: Employment. Rothenburger:Biotest AG: Employment. Wartenberg-Demand:Biotest AG: Employment. Haeder:Biotest AG: Employment. Anderson:Gilead: Consultancy; Sanofi Aventis: Consultancy; Onyx: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership; Oncopep: Equity Ownership.

Abstract Introduction: With approval of Daratumumab (Dara), targeting CD38 has become an attractive therapeutic option for various B cell cancers including WM. Using preclinical models of WM we investigated CD38 expression patterns, the impact of drug-resistance and the anti-WM effects of Dara, in vitro. An important aspect of our evaluation was the identification of optimal CD38 expression that correlates with maximal cytotoxic effects of Dara; such an approach has not been attempted previously in WM. Methods: WM cell lines (BCWM.1, MWCL.1 and RPCI.WM1) with isogenic proteasome inhibitor-resistant (CR, resistant to carfilzomib, bortezomib and ixazomib) and ibrutinib-resistant subclones (IR) were used. CD38 expression was measured by flow cytometry and a grading system was developed based on increasing CD38 MFI values with 1+= <50,000, 2+ = >50,000 and 3+ = >100,000. Daratumumab-induced specific lysis due to antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target ratio (E:T) of 100:1. Peripheral blood mononuclear cells (PBMCs) and complement-containing serum were obtained from healthy human donors. Daratumumab, carfilzomib and ibrutinib were obtained from commercial sources. Results: Expression of CD38 (MFI) was highest in RPCI-WM1 (91295.6) > BCWM.1 (13865.3) > MWCL-1 (9804.19) (Figure 1A.). Intriguingly we observed an interesting change in CD38 expression with induction of drug resistance; all CR clones showed a decrease in MFI (RPCI-WM1/CR; øMFI 55960.5 > MWCL-1/CR; øMFI 3890.0 > BCWM.1/CR; øMFI 1381), while decreased expression was noted in all IR clones except one (MWCL-1/IR; øMFI 6272.96, BCWM.1/IR; øMFI 3076 vs. RPCI-WM1/IR; _MFI 461784.6) (Figure 1B). When we applied our CD38 expression grading system, RPCI-WM1/IR cells classified as Grade 3+, RPCI-WM1 and its CR clones as Grade 2+, while all other WM cells were Grade 1+. We noted that utilization of this grading system was helpful in correlating the extent of cytotoxicity to the level of CD38 expression (Table 1). We next treated WM cells with Dara and observed direct lysis in only RPCI-WM1/CR (43%) > BCWM.1/CR (19%) and RPCI-WM1 (11%) cells. However, when Dara-treated WM cells were cocultured with PBMCs, significantly increased ADCC was noted in RPCI-WM1 (90.3%), RPCI-WM1/IR (88.3%) and RPCI-WM1/CR (68.0%) cells (Figure 1C). Although CDC has been reported as a mechanism of mAb-mediated cell death in WM our evaluation of CDC as a potential effector mechanism in daratumumab-treated WM was negative as expected, highlighting ADCC rather than CDC as the primary cytotoxic mechanism. Conclusion: Our analysis of CD38 in WM preclinical models highlights several important findings: 1.) Expression of CD38 changes as WM tumor clones evolve with induction of drug-resistance; an effect that may also be cell-type specific as evidenced by increased CD38 expression in RPCI-WM1/IR cells, 2.) Daratumumab can exert a direct lethal effect in WM cells but this effect is variable and cell line dependent, 3.) Expression of CD38 is an important factor directing cytotoxicity of Dara however antigen density appears to be an important factor and a grading system can be beneficial for future preclinical and/or clinical studies, 4.) ADCC and not CDC appear to be the prominent mechanism of action and 5.) ADCC-mediated cytolytic activity of Dara is contingent on CD38 expression as evidenced by the most specific lysis being noted in CD38 Grade 2+ and 3+ expressing WM cells (Table 1.). These observations can help direct optimal design of clinical studies with Dara in WM where drug resistance and CD38 expression should be further explored as factors contributing to the clinical efficacy of this new agent. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Abstract Background: High risk CLL patients treated with novel targeted therapies continue to experience disease progression (PD), and more than 50% of these PD patients evolve into Richter's syndrome (RS), (Maddocks, Jama Oncol 2015). Standard chemotherapy for large cell lymphoma has limited efficacy (median overall survival <12 months) in RS. Novel immunotherapy using a PD-1 antibody to block immune checkpoint signals to activate the cytotoxic T -cell has significant efficacy in relapsed Hodgkin lymphoma (Ansell, NEJM 2015). We conducted a phase 2 trial to test the safety and clinical efficacy of the anti-PD-1 antibody pembrolizumab in patients with relapsed/refractory CLL and relapsed low grade B non-Hodgkin lymphoma (B-NHL) (MC1485 trial). Methods: MC1485includes two arms: patients with relapsed/refractory CLL (including RS) and patients with relapsed B-NHL. The main end points of this study are safety and overall response rate. The first 6 patients enrolled in either arm were included in a designed safety interim analysis. Here we report the initial interim analysis from the CLL arm. Relapsed/refractory CLL including RS who had ECOG performance 0-2 and normal organ function were included. Patients with active autoimmune disease or interstitial lung disease were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks until progression, excessive toxicity, or completion of 2 years of therapy. Soluble plasma PD-L1 levels were measured in these 6 patients using an established ELISA assay (Frigola, Clin Cancer Res, 2011). CD8+ T cell subsets were assessed for their functional status using an established analysis (Liu, Oncoimmunology. 2013) at baseline and after 2 cycles of therapies. Results: As of July 27th, 2015, 16 relapsed/refractory CLL patients including 5 RS patients were enrolled. The median age was 71 years (58-81). The median number of prior therapies was 3 (1-6). All CLL patients had received chemoimmunotherapy (FCR/PCR). 4 out of 5 RS patients had received anthracycline-containing chemotherapy. 5 out of 8 patients who had received prior ibrutinib therapy progressed on ibrutinib. 8 patients had 17p-/TP53 mutation. Herein, we report preliminary results from the interim analysis of CLL arm including 4 RS and 2 CLL as well as the 5th RS who had response evaluation by PET. The median cycles of therapy administered was 4 (3-7). One patient experienced grade 2 cytokine release syndrome with transient febrile neutropenia, grade 3 headache and skin rashes. These symptoms resolved with steroid therapy. One patient experienced grade 3 myalgia and was also treated with steroids. The other patients in this safety cohort tolerated pembrolizumab well. The most common drug-related adverse events were grade 1 dyspnea (33%, 2/6) and grade 1 anemia (33%, 2/6). Based on investigator assessment using the Cheson 2007 International Working Group Criteria, 4 out of 5 RS patients had responded to therapy. One patient had a complete remission after 2 cycles and remains in remission. One patient had almost complete PET response after two cycles and is classified as PR as her bone marrow have not been re-evaluated yet. Two patients had notable responses with nodal and skin lymphoma improvements before they showed potential evidence of progressive diseases. The remaining 1 RS and 2 CLL had stable disease and continued on therapy at the last follow-up. Soluble PD-L1 levels (sPD-L1, n=6) were measured at baseline and after 2 cycles of therapies. 3 RS patients who had responded to therapy had decreased or stable sPD-L1 levels. The 4th RS and two CLL had increased sPD-L1 levels and had not demonstrated clinical response. CD8 T-cell subset analyses performed blindly (n=4) showed increased CD8 T cell function in one of the 4 patients tested. This patient who demonstrated CD8 T cell functional enhancement is the only patient in the 4 tested patients who had notable response to pembrolizumab. Conclusion: Pembrolizumab is tolerated in relapsed CLL and RS patients. Early efficacy observed in heavily pretreated RS patients including patients progressed on both anthracycline-based treatment and ibrutinib indicate PD-1 inhibition is a promising novel approach in patients with RS. Our CD8 T cell subset analysis may be used as a biomarker assay to predict clinical response in the era of novel immunotherapy. This trial and further correlative analysis is ongoing and will be presented at the ASH meeting. Disclosures Ding: Merek: Research Funding. Shanafelt:Cephalon: Research Funding; Pharmactckucs: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding; Celgene: Research Funding; Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding. Kay:Tolero Pharma: Research Funding; Hospira: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: Research Funding.

Abstract Background: Aberrant B-cell receptor (BCR) and MYD88 signaling cooperatively drive Waldenstrom macroglobulinemia (WM) tumor cell survival. To sustain BCR-driven growth and maintain optimal cellular homeostasis, WM cells upregulate proteasome function. We have recently described the role of two distinct 19S proteasome-associated deubiquitinating enzymes (DUBs), USP14 and UCHL5, in maintaining WM-cell viability. Pharmacologic inhibition with b-AP15 (commercial compound) results in WM-cell death and accumulation of high molecular weight ubiquitinated protein conjugates; an event that is phenocopied by siRNA knockdown of USP14 and UCHL5. VLX1570 is a lead-optimized clinical grade analog of b-AP15 with enhanced solubility, stability and target residence time. We investigated the effect of VLX1570 on BCR components, WM cell survival proteins and its in vivo activity in a human xenograft model of WM. Materials: VLX1570 was obtained from Vivolux AB and bortezomib from Sellekchem. Human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1) and their drug-resistant derivatives were used (total n=12). Bone marrow aspirates from WM-patients and from healthy donors were obtained from the Mayo Clinic biospecimens core. PBMCs from healthy donors were used as comparators where needed. NOD/SCID mice were obtained from Taconic Labs and used for profiling the in vivo properties of VLX1570. Results: We first examined for USP14/UCHL5 protein expression by immunohistochemical analysis in primary tumor tissue from WM patients and compared it to healthy donor cells. WM patient-derived cells exhibited greater staining intensity of both DUBs. Preferential binding of VLX1570 to USP14 and UCHL5 in WM cells was probed in a WM cell using an enzyme-based competition assay (HA-Ub-Vs), which demonstrated time-dependent decrease in labeled USP14 and UCHL5 after treatment with VLX1570. In drug-resistant (bortezomib or ibrutinib-resistant) WM cells, VLX1570 induced >50% tumor-specific apoptosis and displayed synergistic activity with ibrutinib (median CI: 0.5). In addition to ER stress markers, we had previously observed that gene expression of BCR-signalosome components were reduced in VLX1570-treated WM cells (NanoString platform) and this was substantiated at the protein level where p-SYK, SYK and BTK decreased in a time-dependent manner. BCR end-effectors were probed in response to VLX1570 and a decrease in nuclear translocation of NFkB (p65) was noted. USP14 has been shown to control the availability of the chemokine receptor CXCR4, which regulates homing of WM cells to bone marrow. After VLX1570 treatment, we noted a significant decrease in CXCR4 surface receptor expression in all WM cell lines tested and primary WM cells. Lastly, to determine the in vivo anti-WM effects of VLX1570, we injected NOD/SCID mice (xenotransplanted with RPCI-WM1-Luc cells) with VLX1570 (I.P. route, 4.4mg/kg every other day) and measured tumor burden along with pharmacodynamic indicators. VLX1570-treated mice (n=7) demonstrated significantly lower tumor volumes by caliper measurement and bioluminescent signal (p=0.01 - 0.003) as well as reduced serum human-IgM, compared to vehicle-treated mice (n=7). Importantly, mice treated with VLX1570 survived longer vs. vehicle-treated mice (median survival, 62 vs. 44 days, respectively, p=0.0008). No unforeseen toxicities were noted and weight of the mice in vehicle vs. VLX1570-treated mice did not significantly change. IHC analysis in mice tumor tissues demonstrated reduced CXCR4, human-IgM, Ki67 and increased cleaved caspase-3 and ubiquitinated (k48-linked) protein level, the latter serving as a more specific indicator of USP14/UCHL5 inhibition. Conclusions: We provide substantial in vitro and in vivo evidence on the anti-WM effects of a novel first-in-class clinical grade USP14/UCHL5 inhibitor, VLX1570. In addition to anticipated induction of ER stress machinery, we describe for the first time VLX1570-mediated modulation of BCR components and WM cell survival proteins as well as demonstrate its robust activity in an aggressive human xenograft model of WM. These data provide the rationale for a clinical trial of single agent VLX1570 in patients with symptomatic WM. Disclosures Martin: Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Gullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models. Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000. Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S). Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Abstract Background: Current tumor profiling analytics provide some insight into the various molecular abnormalities and their individual consequences on oncogenic signaling. However, these analyses are limited by their lack of integration where the combined effect of individual mutations, gene copy number variations and chromosomal aberrations are not consolidated to create the global molecular architecture that supports neoplastic growth, particularly in the context of drug resistance. Consequentially, identities of the preferential oncogenic pathway(s) tumor cells employ to oppose the effects of targeted therapies remain cryptic and unactionable. Here we present a simulation-based method, which not only replicates the molecular architecture of ibrutinib-resistant Waldenstroms Macroglobulinemia (WM, for which ibrutinib is the only FDA-approved agent) in silico, but also predicts cell sensitivity towards existing drugs, which we validated experimentally for potential clinical translation. Materials: We used the newly established human WM cell line, RPCI-WM1/IR, as a surrogate model of ibrutinib-refractory WM. Genomic data including whole exome sequencing (WES) and copy number analysis (CNA) was utilized for the creation of an avatar of RPCI-WM1/IR, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were nominated. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of the WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. Results: Several genomic aberrations were used to create the RPCI-WM1/IR simulation avatar. Functional activity (based on mutation or copy number alteration) of several ibrutinib targets or transcription factors associated with BTK activity such as FYN, SP1, BMX and FRK were predicted to be lost. Increased expression of CAV1, which also inhibits BTK mediated signaling, was increased. An increase in CSNK2B, which activates PU.1- a transcriptional target of BTK, was also observed. Of note, no CXCR4 mutations, which have been shown to impact ibrutinib response, were observed. Next, the cytotoxic potential of over 150 FDA approved drug (and some in experimental stages) were simulated individually and in combination on the RPCI-WM1/IR avatar. In silico modeling predicted aberrant activity of aurora kinase A (AURKA) and its associated signaling partners, which could be disrupted with the (AURKA) inhibitor, tozasertib. AURKA activation was predicted as upregulated due to alterations in several genes: RASA1 loss and SOS1 increase --> increased ERK --> increased ETS1 --> increased AURKA. High beta-catenin signaling (high CTNNB1 and FZD1/4 and low AXIN1 and GSK3B) was also shown to increase AURKA. The simulation predictions were experimentally validated in vitro where AURKA inhibition with tozasertib significantly inhibited proliferation of RPCI-WM1/IR cells (IC50~14nM) as well as inducing apoptosis (48hr, 20nM treatment) and cell-cycle arrest. Conclusions: Our data demonstrates the potential of in silico modeling in predicting novel drug targets, allowing guidance in 1.) Delineating operational oncogenic circuits in an ibrutinib-resistant state by reanimation of the molecular architecture in silico, 2.) Calculating the impact of individual genomic abnormalities and their collective influence on maintaining tumor survival and 3.) Performing a rapid in-silico drug-sensitivity screen directed by the pathway analyses, which can be validated experimentally using standard assays. This novel approach holds tremendous potential in creating highly personalized therapies for ibrutinib-refractory WM patients based on unique genetic signatures. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

Abstract Abstract 3031 Background: Treatment-induced peripheral neuropathy (TIPN) can be a debilitating side-effect as well as a therapy-limiting complication in multiple myeloma (MM). Thalidomide (THAL) and bortezomib (BTZ) are two therapies frequently associated with TIPN in MM. Carfilzomib (CFZ) is a novel and highly selective epoxyketone proteasome inhibitor that differs from BTZ both structurally and mechanistically. CFZ overcomes BTZ-resistance in vitro, lacks the off-target activities of BTZ in preclinical studies, and does not cause neurotoxicity in long-term (6–9 month) chronic animal toxicology studies (Kirk et al. Blood, 2008). Single-agent CFZ produces durable responses in relapsed or refractory (R/R) MM without dose-limiting PN. Here we report on the clinical experience with single-agent CFZ in the Ph 2b PX-171-003-A1 trial in patients (pts) with R/R MM and Grade (G) 1/2 PN at study entry. Methods: Pts received CFZ at 20 mg/m2 IV, on Days 1, 2, 8, 9, 15, and 16 in a 28-day cycle (C) for the first C followed by 27 mg/m2 thereafter for up to 12 C. Pts completing 12 C were eligible to enter an extension study. Responses and progression were determined according to the International Myeloma Working Group (IMWG) criteria and were assessed by an Independent Review Committee (IRC). PN data were collected for all pts on study and included neuropathy history, neurological physical exam and PN-related quality of life data (FACT-GOG/NTx v 4.0 scores) collected at screening. Prospective neurological exams and subjective reporting of PN occurred every 2 cycles until study discontinuation to proactively assess for PN. Adverse event (AE) data were also collected, with AEs reported as ‘neuropathy peripheral', ‘neuropathic pain', ‘neuropathy', and ‘peripheral sensory neuropathy' included as PN. AE reports of ‘paraesthesias' and ‘dysesthesias' were counted separately. Results: Of the 266 pts with R/R MM in PX-171-003-A1, 237 (89%) had a history of PN which was attributable to prior anti-myeloma therapy, including THAL (108 pts/41%), BTZ (134 pts/50%), or both BTZ and THAL (17 pts/6.4%). 206 of the 266 (77%) had G1/2 PN at baseline and a median disease duration of 5.9 years. This subset with active PN at baseline had received a median of 5 prior lines of therapy (range 1–20), with a median 13 anti-myeloma agents, and a median of 2 prior BTZ- and 1 prior THAL- containing regimens. Prior therapies included 100% BTZ, and 100% either THAL (77%) or prior lenalidomide (95%). Responses in the subset of pts with baseline PN were nearly identical to those seen in the full study population with an overall response rate (ORR; ≥ partial response [PR]) of 24% and a clinical benefit response rate (CBR; ≥ minimal response [MR]) of 36%. The median duration of response (≥PR) was 7.4 mo (95% CI 5.6–not reached) and median duration of MR was 6.3 months in both the overall and PN-baseline cohorts. OS and TTP data will also be reported. The most common treatment-emergent ≥G3 adverse events regardless of relationship to study drug were primarily hematologic and were as follows: thrombocytopenia (24%), anemia (21%), lymphopenia (11%), pneumonia (9%), neutropenia (9%), fatigue (7%), hypercalcemia (7%), and hyponatremia (6%). Although 77% of pts had G1/2 PN at baseline, new onset PN was infrequent with PN AEs of any grade reported in 31 (15%) pts and G3 PN reported in only 1 (0.4%) pt. New onset or worsening of paraesthesias (6.8%) and dysesthesias (0%) was also infrequent. Conclusions: Analysis of the subset of pts (77%) with active PN (G1/2) in this single-agent Ph 2 trial of CFZ in pts with R/R MM demonstrated that PN has no impact on depth or durability of responses, or on the tolerability of CFZ, in heavily pretreated pts with multiply relapsed and refractory MM. Reports of new or worsening PN were very uncommon, and paraesthesias and dysesthesia were generally infrequent and mild. CFZ can be given to pts with baseline PN with little risk of exacerbation; prolonged therapy is possible in this population. Disclosures: Martin: Celgene: Honoraria; Onyx: Consultancy. Singhal:Celgene: Speakers Bureau; Takeda/Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Onyx: Research Funding. Vij:Onyx: Honoraria. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Stewart:Millennium: Consultancy; Celgene: Honoraria. Jagannath:Millenium, OrthoBiotec, Celgene, Merck, Onyx: Honoraria; Imedex, Medicom World Wide, Optum Health Education, PER Group: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lonial:Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Jakubowiak:Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Centocor Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kukreti:Celgene: Honoraria; Roche: Honoraria; Ortho Biotech: Honoraria. Alsina:Millenium: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy. Zonder:Millenium: Consultancy, Honoraria, Research Funding; Cephalon: Research Funding; Celgene: Honoraria. Wong:Onyx Pharmaceuticals: Employment. Vallone:Onyx Pharmaceuticals: Employment. Chang:Onyx Pharmaceuticals: Employment. Kauffman:Onyx Pharmaceuticals: Employment. Siegel:Millenium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.